[A new method of producing biologically active nanocomplexes by non-covalent conjugation of proteins with viral particles].

Currently, a range of biologically active molecules have been attached to plant and bacterial viras nanoscaffolds, yielding stable nanoparticles that display multiple copies of the desired molecule. In this paper we propose a new method of non-covalent attachment of peptides to the surface of virios. We have demonstrated that this method is efficient in a model system that includes tobacco mosaic virus particles, synthetic polycation (quaternized poly(4-vinylpyridine) carrying ethyl ethyl pendant radicals) and polypeptide of interest. This principle of step-by-step binding to the surface of virions was used for electrostatic association with hydrophilic fragment of influenza virus haemagglutinin.

Fibrinogen is an antioxidant that protects beta-lipoproteins at physiological concentrations in a cell free system.

Oxidation of beta-lipoproteins has been linked to the development of arteriosclerosis. Using a copper mediated cell free system to oxidize beta-lipoproteins, we found that beta -lipoproteins isolated from plasma were less susceptible to oxidation than lipoproteins from serum and that this was probably due to inhibition by fibrinogen, because removal of fibrinogen from plasma enhanced oxidation, while addition of fibrinogen restored inhibition. Fibrinogen inhibited conjugated diene formation and peroxide formation assayed by the xylenol orange assay (absorbance+/-confidence interval: 0.155+/-0.007 with fibrinogen vs 0.255+/-0.014 without) and retarded copper mediated oxidation of apolipoproteins in low density lipoproteins, reducing the distance of electrophoretic migration by 5 mm. The effect of fibrinogen was not due to chelation of copper, since it provided protection when hydrogen peroxide was substituted for copper as an oxidizing agent. At normal physiological concentration equivalents, fibrinogen showed superior antioxidant properties compared to albumin, melatonin, vitamin C and vitamin E and was superior to the vitamins when compared on an equimolar basis. Other studies have shown the fibrinogen to be more oxidizable than other major plasma proteins and to inhibit peroxide production. Because of its high mass concentration, we postulate fibrinogen is an important antioxidant protecting beta-lipoproteins in plasma and that it may be important in protecting lipoproteins in tissue spaces.

Adenosine induced atrial fibrillation precipitating polymorphic ventricular tachycardia.

An 86-year-old female developed supraventricular tachycardia 36 hours after a myocardial infarction (MI). She developed atrial fibrillation and polymorphic ventricular tachycardia (PVT) following administration of 12 mg of adenosine. The PVT caused hemodynamic instability with no response to cardioversion, but termination with procainamide. The heart is vulnerable to hemodynamically unstable, possibly lethal, PVT early after MI under some circumstances. This vulnerability may be exposed following administration of adenosine. Extra caution is warranted when using adenosine in the post-MI period.

Simpler technique for measuring oxidative susceptibility by heparin affinity column isolation of lipoproteins.

Variations in the in vitro oxidative susceptibility or resistance of lipoproteins have been used to test the effect of ingested antioxidants and may prove to be a marker for coronary artery disease. Here we describe a simple technique for isolating and oxidizing beta-lipoproteins that may have utility in the clinical laboratory. Electrophoretic profiles showed that beta-lipoproteins were separated from alpha-lipoproteins and essentially from most other serum proteins using heparin affinity columns. Lipoproteins were normalized in the reaction mixture by measuring apo B in the beta-lipoprotein eluate using an automated apo B method, which gave good recoveries of 106-112%. Copper mediated oxidation was monitored by measurement of conjugated dienes formation at 234 nm for 300 min. When the reaction mixture included beta-lipoprotein eluate containing 0.03 g/L of apoB and 5 micromol/L copper sulfate, conditions were effective for obtaining complete oxidation while allowing reproducible measurements, with between day coefficients of variation of 2.35%, 14.6%, and 10.5% for lag, propagation and plateau phases, respectively. Beta-lipoproteins isolated from serum were more susceptible to oxidation than beta-lipoproteins from plasma apparently due to inhibition of oxidation by fibrinogen which co-eluted with beta-lipoprotein from plasma. For this reason, we recommend using serum preserved with EDTA for this assay.

When is a heterophile antibody not a heterophile antibody? When it is an antibody against a specific immunogen.

Heterophile antibodies are antibodies produced against poorly defined antigens. These are generally weak antibodies with multispecific activities. Human anti-animal antibodies that develop as a result of treatments with animal immunoglobulins are antibodies with strong avidities, produced against well-defined antigens. Although heterophile antibodies and human anti-animal antibodies interfere with immunological assays by similar mechanisms, modes for identifying the sources of the antibodies and for circumventing or retarding the interference may differ. Unfortunately, there has not been a well-organized attempt to encourage correct definition of these antibodies. This problem of inexact definition is highlighted by recent articles in this Journal. In the present discussion, we examine the history leading to this problem and discuss the origins and the reasons that the nature of the antibody is important for rectifying the problem. We propose a simple nomenclature for general usage that should appropriately characterize these antibodies in most cases.

Fos expression during the critical period in visual cortex: differences between normal and dark reared cats.

The present study tested further the notion that immediate early gene expression is involved in neuronal plasticity during the critical period of visual cortex (VC) by comparing Fos induction in normal and dark reared (DR) cats. Western blots indicated that the level of induced Fos expression was higher in normal than DR cat VC at 5 weeks of age, comparable at 10 weeks, and higher in DR than normal cat VC at 20 weeks. Immunohistochemistry indicated that at 5 weeks Fos was induced in cells of all VC layers in both rearing conditions, but to a greater extent in normal than DR cats. At 20 weeks, Fos was largely restricted to cells above and below layer IV in both rearing conditions, but was induced to a greater extent in DR than normal cats. Thus, dark rearing appears to have very similar effects on Fos expression as it has on neuronal plasticity during the postnatal critical period.

Sustained improvement in flow-mediated vasodilation after short-term administration of dobutamine in patients with severe congestive heart failure.

BACKGROUND: In patients with severe congestive heart failure (CHF), short-term administration of dobutamine exerts sustained clinical benefits that are partially mediated by a training-like effect on skeletal muscle. Recently, physical training has been shown to enhance endothelial function in the skeletal muscle vasculature by improving endothelial function. Whether the dobutamine-induced training effect is also associated with an improvement in endothelial function in the skeletal muscle vasculature is currently unknown. METHODS AND RESULTS: Flow-mediated vasodilation in response to peak reactive hyperemia was evaluated in the forearms of 9 patients with severe CHF who were treated with dobutamine for 72 hours. Resting and peak hyperemic brachial artery blood flow and diameter (BABF [mL/min] and BAD [mm], respectively) were measured by 2-dimensional and Doppler ultrasonography at baseline, at 3 and 72 hours during dobutamine infusion, and at 2 and 4 weeks after discontinuation of dobutamine therapy. In addition, the brachial artery response to sublingual (SL) administration of nitroglycerin (NTG) was evaluated at baseline and at 2 and 4 weeks after discontinuation of dobutamine therapy. Ten patients with severe CHF who did not receive dobutamine served as control subjects. Resting BABF was significantly increased at 3 and 72 hours (391.2+/-31.8 and 366.8+/-31.0 mL/min, respectively, compared with 289.8+/-18.6 mL/min at baseline; P<0.05). Peak hyperemic BABF was not altered by dobutamine infusion compared with baseline values. The increase in BAD during peak hyperemic response was greater after infusion of dobutamine for 72 hours (15.2+/-2.7% versus 9.1+/-1.8%, P<0.05) and remained significantly greater for >/=2 weeks after discontinuation of dobutamine (12.3+/-2.2% versus 9.1+/-1.8%, P<0.05). In contrast to the peak hyperemic response, the increase in BAD (%) induced by SL NTG was unchanged by administration of dobutamine for 72 hours. Two and 4 weeks after discontinuation of dobutamine, NTG-induced increases in BAD were similar to the BAD noted at baseline. CONCLUSIONS: In patients with severe CHF, short-term administration of dobutamine for 72 hours selectively improves vascular endothelial function for >/=2 weeks.

Mechanized lipoprotein(a) assay as a marker for coronary artery disease illustrates the usefulness of high lipoprotein(a) levels.

Only a few simple lipoprotein(a) [Lp(a)] assays are available in kit form for use in clinical laboratories. The present study compares the analytical and clinical performance of a mechanized immunonephelometric method to enzyme-linked immunosorbent assay. Clinical performance was evaluated by measuring lipoprotein markers in 191 patients, with the extent of stenosis defined by angiography. Analytically, both methods showed little or no correlation with cholesterol, high density lipoprotein cholesterol, elevated triglycerides, apo A-I and apo B, while they showed good agreement with one another (r = 0.88). The methods showed comparable well known differences between black and white persons. Logistic regression indicated that Lp(a) was a weak but independent marker for coronary artery disease (CAD). Receiver operator characteristic curve analysis showed an association with CAD only at higher Lp(a) concentrations. We conclude that Lp(a) at higher concentrations may be a contributory marker for CAD and that mechanized nephelometric assays for it can be used in the clinical laboratory.

Expression of two forms of glutamic acid decarboxylase (GAD67 and GAD65) during postnatal development of the cat visual cortex.

The postnatal development of GAD67 and GAD65 protein expression and of GAD67 positive neurons and GAD65 containing axon terminals in cat visual cortex was studied. Western blot analysis showed that the expression of both GAD67 and GAD65 increased to approximately two-thirds of the adult level during the first 5 postnatal weeks and gradually increased thereafter. In adult cats, immunohistochemistry showed that GABA and GAD67 containing neurons were found in all cortical layers. Faint cell body staining was seen with the antibody to GAD65, but it densely labeled puncta. In neonates, GABA and GAD67 immunoreactivity was most intense in two distinct bands, one superficial (Layer 1/Marginal zone), another deep (Layer VI/Subplate). Unlike in adults, GAD65 positive cell bodies were clearly evident in neonates and distributed similarly to, but less frequently than, GABA and GAD67. These GAD65 positive cells frequently had morphologies suggestive of embryonic cells and largely disappeared in older animals. During postnatal development, the neurochemical differentiation of GAD67 positive neurons and GAD65 positive axon terminals across visual cortical laminae followed an inside-outside developmental pattern, which reached adult levels after 10 weeks of age. These results suggest that postnatal development of the visual cortical GABA system involves three distinct processes: (A) a dying off of embryonic GABA cells which could play a role in formation of the cortical plate; (B) a period of relative quiescence of the VC GABA system in the first 5 postnatal weeks which could maximize excitatory NMDA effects during the rising phase of the critical period; (C) the prolonged postnatal maturation of the adult GABA system which could be involved in the crystallization of adult physiological properties and the disappearance of neural plasticity.

Immediate early gene expression in cat visual cortex during and after the critical period: differences between EGR-1 and Fos proteins.

Immediate early gene (IEG) expression in the cat visual cortex is highly responsive to visual input and may initiate genetic mechanisms responsible for neuronal plasticity. The present study used immunohistochemical methods to address two issues regarding IEG expression in response to visual input. One was to define the differential response of distinct IEG families by comparing EGR-1 (also termed zif-268, NGFI-A, and Krox-24) and Fos proteins. The second was to determine whether IEG expression, in addition to reflecting neural activity, is related to the state of plasticity by comparing young and adult visual cortex. Immunoreactivity of the two IEG proteins was compared between 5-week-old and adult cats under three conditions of visual input: ambient light to assess basal levels of expression, 1 week of darkness to assess the effect of reduced activity, and exposure to light after 1 week of darkness to determine rapid changes in expression as a result of visual input. At both ages, there were marked differences in the expression of the two IEG proteins. EGR-1 responded to visual input with sustained changes in its level of expression. It showed high basal levels, reduced expression in darkness, and a rapid return to high constitutive levels with the introduction of light. Fos showed a markedly different profile. It had very low basal expression which was not demonstrably affected by darkness and its principal response was a marked transient induction upon exposure to light after darkness. These unique changes in expression highlight the complex response across IEGs to environmental input and suggest a genetic "on/off' signaling mechanism. There were marked differences in the laminar distribution of EGR-1 and Fos proteins between young and adult cats. In young animals, cells in all visual cortical layers showed high levels of EGR-1 and Fos proteins. In adults, immunostaining was largely specific to cells located above and below layer IV and only very faint labeling occurred within layer IV. These differences in laminar distribution between ages are inconsistent with a simple explanation of IEG expression in terms of neural activity level; rather, they suggest a relation between IEG expression and the state of plasticity in visual cortex.

Developmental expression of the immediate early gene EGR-1 mirrors the critical period in cat visual cortex.

Immediate early gene (IEG) expression in the central nervous system is thought to play a role in coupling extracellular stimulation with the transcriptional events responsible for long-term functional changes in neurons. The goal of the present study was to determine the postnatal developmental profile of EGR-1 protein (also termed zif268, Krox-24, NGFI-A) expression across the layers of cal visual cortex and relate it to the state of visual cortical development and plasticity. Using a polyclonal antibody, EGR-1 immunoreactivity was studied in animals of various postnatal ages (from 0.5 week to adult). In very young animals (0.5 weeks), EGR-1 positive cells were restricted to deep cortical layers (layer VI/Subplate). With the increasing age, EGR-1 immunoreactivity spread across layers of the visual cortex in an inside-outside manner, and by 5 weeks of age, EGR-1 protein was highly expressed in all layers. EGR-1 expression remained high until approximately 10 weeks of age and then gradually began to decline in layer IV with little change in supra- and infragranular layers. In adult animals, EGR-1 was located predominantly in the layers above and below layer IV. This pattern of EGR-1 expression in developing cat visual cortex has both temporal and laminar similarities with the development of visual cortical connectivity, with the development of orientation selective receptive field properties, and with the level of visual cortical plasticity, suggesting an involvement of EGR-1 expression in these processes.